On The Identification of Body Fluids and Tissues A Crucial Link in The Investigation and Solution of Crime.
On The Identification of Body Fluids and Tissues A Crucial Link in The Investigation and Solution of Crime.
On The Identification of Body Fluids and Tissues A Crucial Link in The Investigation and Solution of Crime.
T A C G
G C A T
genes
Review
On the Identification of Body Fluids and Tissues: A Crucial
Link in the Investigation and Solution of Crime
Titia Sijen 1,2, * and SallyAnn Harbison 3,4
1 Division Human Biological Traces, Netherlands Forensic Institute, Laan van Ypenburg 6,
2497 GB The Hague, The Netherlands
2 Swammerdam Institute for Life Sciences, University of Amsterdam, Science Park 904,
1098 XH Amsterdam, The Netherlands
3 Institute of Environmental Science and Research Limited, Private Bag 92021, Auckland 1142, New Zealand;
[email protected]
4 Department of Statistics, University of Auckland, Private Bag 92019, Auckland 1142, New Zealand
* Correspondence: [email protected]; Tel.: +31-70-888-6666
Abstract: Body fluid and body tissue identification are important in forensic science as they can
provide key evidence in a criminal investigation and may assist the court in reaching conclusions.
Establishing a link between identifying the fluid or tissue and the DNA profile adds further weight
to this evidence. Many forensic laboratories retain techniques for the identification of biological
fluids that have been widely used for some time. More recently, many different biomarkers and
technologies have been proposed for identification of body fluids and tissues of forensic relevance
some of which are now used in forensic casework. Here, we summarize the role of body fluid/ tissue
identification in the evaluation of forensic evidence, describe how such evidence is detected at the
crime scene and in the laboratory, elaborate different technologies available to do this, and reflect real
life experiences. We explain how, by including this information, crucial links can be made to aid in
the investigation and solution of crime.
Citation: Sijen, T.; Harbison, S. On
the Identification of Body Fluids and
Keywords: forensic; review; body fluid; organ; tissue; identification; mRNA; DNA methylation;
Tissues: A Crucial Link in the
Investigation and Solution of Crime.
activity level
Genes 2021, 12, 1728. https://doi.org/
10.3390/genes12111728
Academic Editor: Niels Morling 1. Activity-Level Evaluations: The Context of Body Fluid and Tissue Identification
Body fluid and tissue identification can add evidence in criminal investigations by
Received: 12 October 2021 establishing a crucial link between the donor, the cell type and the activities that occurred.
Accepted: 26 October 2021
In this review, we first provide the forensic context of body fluid and tissue identification
Published: 28 October 2021
which resides in activity-level evaluations, then we discuss methods to locate body fluids
at the scene or in the laboratory, which is followed by the major marker methodologies to
Publisher’s Note: MDPI stays neutral
analyze them. One of these methods is mRNA profiling, and we take a retrospective look
with regard to jurisdictional claims in
at casework details and verdicts for mRNA cases in the Netherlands. This leads us to a
published maps and institutional affil-
criminalistic view on targets that can be accommodated in assays and the interpretation of
iations.
cell typing results for instance, in mixed stains, which needs a very different approach from
the interpretation of DNA results. Lastly, we consider possible other marker types that
have been suggested, and practical aspects when introducing cell typing into a forensic
laboratory.
Copyright: © 2021 by the authors.
Licensee MDPI, Basel, Switzerland. 1.1. Prosecution and Defense Scenario
This article is an open access article
Increasingly, cases come to court in which the presence of cellular material of a person
distributed under the terms and
is not disputed but the activity that caused the deposition is. The debate then centers around
conditions of the Creative Commons
the question ‘how did his/her DNA get there’? Forensic scientists approach such questions
Attribution (CC BY) license (https://
by performing activity-level evaluations [1–8]. Figure 1 shows activity-level evaluation in
creativecommons.org/licenses/by/
4.0/).
the context of forensic human biology analyses. Generalizing, activity-level evaluations
Figure 1. Stain identification in the context of the forensic process showing the link between ‘who’,
Figure 1. Stain identification in the context of the forensic process showing the link between ‘who’,
‘what’ and and
‘what’ ‘how’.
‘how’.
The ‘findings’ in the case can relate to cellular material matching victim or suspect
1.2. Addressing
which underAlternative Scenariosof an offensive activity (e.g., blood matching the victim on
H1 is indicative
Forensic
the blade studies
of a knifeaim to develop
indicative approaches
of the to assist
victim being in addressing
stabbed; offensive
cellular material and al- the
matching
ternative scenarios. To address deposition during the offense or during an earlier or later out
suspect on the handle of a knife supporting the proposition that the suspect carried
the stabbing).
encounter, inferringUnder H2,since
the time this deposition
questioned(TSD)cellular
may material on the
be helpful. Forevidentiary
bloodstains,item
var-may
ious result from a number
spectroscopic of scenarios:
and chemometric TSD (1)methods
the cellular
havematerial was deposited
been proposed that relynot during the
foremost
offense to
on changes buthemoglobin
at an earlier[9–13].
or later
Antime (the suspect
approach did not
that would be stab but picked
applicable to moreup body
the knife
after
fluids thantheblood
stabbing); (2) the
is based cellular material
on differences in thewas not deposited
degradation by direct
over time contactRNA
of distinct but was
molecules outside the human body. Determining relative expression ratios may allow in- the
the result of secondary (or tertiary or further) transfer (the suspect shook hands with
realof
ference perpetrator
TSD, although who robustness
then handled theyet
is not knife in the
at the stabbing);
level needed (3)
for the item casework
forensic on which the
[14–19]. Recently, microbes were explored for TSD purposes of saliva samples withoffense
cellular material was deposited belongs to the suspect but was used in the some by
another
success, person
but the high(the knife (ownedvariation
inter-individual by the suspect who uses
can surpass the it to cut food) was
time-dependent handled by
variation
[20].the real perpetrator in the stabbing); (4) the cellular material was deposited on the knife in
aTo
different
addressway (the victim
the question of fell into the
primary knife). or secondary transfer, TPPR (transfer,
deposition
prevalence, persistence and recovery) issues are studied [21–31]. The aspect of transfer
1.2. Addressing Alternative Scenarios
Forensic studies aim to develop approaches to assist in addressing offensive and
alternative scenarios. To address deposition during the offense or during an earlier or
later encounter, inferring the time since deposition (TSD) may be helpful. For bloodstains,
various spectroscopic and chemometric TSD methods have been proposed that rely fore-
most on changes to hemoglobin [9–13]. An approach that would be applicable to more
body fluids than blood is based on differences in the degradation over time of distinct
RNA molecules outside the human body. Determining relative expression ratios may
allow inference of TSD, although robustness is not yet at the level needed for forensic
casework [14–19]. Recently, microbes were explored for TSD purposes of saliva samples
with some success, but the high inter-individual variation can surpass the time-dependent
variation [20].
To address the question of primary deposition or secondary transfer, TPPR (transfer,
prevalence, persistence and recovery) issues are studied [21–31]. The aspect of transfer
appears the most complex among TPPR issues and many studies have gained information
on transfer rates, and the various aspects that influence transfer rates such as wet or
Genes 2021, 12, 1728 3 of 32
dried state, application of friction and pressure, smooth or rough surface and size of
the stain [32–38]. Generally, probabilistic frameworks are used to guide the evaluation,
and Bayesian networks are appreciated for their graphical strength in this evaluative
process [39,40].
Inferring the body fluid(s) present in stains is, in the context of secondary (or more)
transfer, important for two reasons: (1) to assess the cellular content of the primary stain in
relation to the DNA recovered from the evidentiary sample as some fluids have very high
cellular content (for example nasal secretion), whilst others contain much fewer cells (for
instance urine) and (2) to assess whether the body fluid involved is likely to occur in what
is suggested) to be the primary location [41]. For example, intimate body fluids (vaginal
cellular material, menstrual secretion, semen) can be found in underpants of the wearer,
but are less expected on hands or touched items [42–44]. Saliva and nasal secretion, on the
other hand, can reside on hands or items, and blood could occur when people have (small)
wounds. Organ tissues (for instance, CNS (central nervous system), heart, or adipose
tissue), on the other hand, are not expected on hands or on items.
The scenario that another person and not the suspect utilized an item (belonging to the
suspect) on which cellular material was deposited in the offense, can relate to objects (e.g., a
knife used for stabbing or a bottle that was inserted vaginally) or clothing/ shoes that
appear to have been worn during the offense for instance because blood spatter or organ
tissue matching the victim found on them. Cell typing of samples from such items may
provide the crucial link of the evidentiary item to the alleged crime (the knife carries muscle
tissue matching the victim on the blade; the bottle contains vaginal material matching the
victim on the bottle neck; the shoe carries CNS tissue matching the head-kicked victim),
but the link to the perpetrator needs to be provided by other means. This may be addressed
for instance through fingerprint analysis (e.g., on the bottom of the bottle or the handle of
the knife), DNA-analysis (e.g., of the shoelaces or the insides of the neck and collar areas of
the sweater) or CCTV-footage or witness reports on the suspect acting in the stabbing or
wearing the shoes or clothing at the time of the attack.
When a different way for the deposition of the cellular material is proposed (e.g.,
shooting out of self-defense, intercourse with consent), the evaluation is often at the offense
level, which means the domain of the court [2,45,46]. With such scenarios, DNA nor cell
typing add value in most of the cases.
mostly presumptive and visual in nature, or in a forensic laboratory (using samples and
items collected at the scene) where more detailed analysis is possible.
visualised two years later, using a light source between 415 nm and 460 nm in combination
with a yellow or orange filter.
To improve the utility of light sources and photography at crime scenes, a study was
conducted [51] combining a blue Crime-lite XL light source in combination with a 360◦
camera to capture semen and saliva stains on a number of different substrates. This study
was carried out in a mock crime scene indoor setting using volunteers to examine the
images and locate the stains. The colour of the substrate was found to have an effect on the
ability to see the stains, however, in general, the combination of light source and camera
was effective.
A side by side comparison of the Crime-lite® 82S, Polilight® PL400 and Polilight®
PL500 [58] was recently carried out, comparing the sensitivity and performance of each
on dilutions of saliva and semen on different fabrics. The LED light source used in the
Crime-lite® 82S provided better detection of dilutions of saliva and overall performed
better as a screening tool.
Recent developments include combined handheld, UV/Vis/IR multi-wavelength
light sources and camera combined, with automatic filter selection all controlled from
an integrated touch screen for example the Crime-lite AUTO. This handheld instrument
appears to offer a number of advantages for crime scene use, the most obvious of which is
the combined handheld unit. See also Section 2.2.3 for an alternative.
These methods can also be employed in a forensic laboratory when transportable items are
involved. In the remainder of this section, we discuss methods that are primarily laboratory-
based for locating biological fluids. These chemical, immunological, and spectroscopic
tests are often used to presumptively identify biological fluids before conducting further,
more specific testing where necessary.
peripheral blood [86]. The alternative modification, external reflection FT-IR spectroscopy,
has also been proposed [59] to specifically identify body fluids associated with sexual
assault. The effects of different fabric colours and commonly used household substances
on identification were also assessed. These instruments are not handheld and, therefore,
not compatible with examinations at the crime scene.
forensic science [121]. The main advantages of MPS is the high multiplexing capacity, that
amplicons can have overlapping (small) sizes and that nucleotide variation is detected.
These first two features are of importance with forensic traces as they are often of minute
amounts and suffer from degradation due to external factors. The forensic importance of
analysing sequence variation in RNA amplicons will be discussed in Section 6.4. Other
techniques for detecting markers are given in Section 3.1.3.
in the identification of semen, saliva, venous blood and menstrual blood in body fluid
mixtures and in crime scene stains [158]. Methylation state specificity was achieved using
the single-base extension primers of the SNaPshot assay. Amplification refractory mutation
system-PCR (ARMS-PCR), otherwise known as allele-based PCR [159], in combination with
CE was used as an alternative method to test 22 possible markers for venous blood, saliva,
semen, menstrual blood, and vaginal cellular material in multiplexes [160]. A random
forest model was employed and performed well in predicting single source body fluids
with high prediction accuracy (99.66%).
The multistep process of bisulfite conversion, PCR and detection by CE or sequencing
is vulnerable to variation due to poor sample quality and quantity and especially degrada-
tion during the bisulfite conversion step. A new multiplex quantitative RT PCR method
to measure the amount of genomic and bisulfite-converted DNA, and the degradation
level of the conversion step as well as the conversion efficiency has been described [161].
Besides, natural variation in methylation status has been found between individuals, and
some tissue-specific differentially methylated regions are susceptible to change due to
environmental factors and age. For example, methylation of a CpG site in PRMT2 in blood
samples was found to be an age-associated marker, whereas no significant difference based
on age was observed for three spermatozoa-specific hypomethylated markers DACT1,
USP49, and PRMT2 in men of different ages [149,152]. Others have demonstrated that
within-person variation in methylation ratios was not observed, but variation was observed
between people in an assay designed to identify vaginal cellular material [157].
Many studies have focused on specific body sites/ fluids for example the microbiomes
of saliva [192,193], faeces [194], the vagina [164,195] with recent publications focusing
on distinguishing between different sources of blood (menstrual, venous, fingerpick and
nasal) [196,197] and semen [198]. Lactobacillus species have been incorporated into mRNA
multiplexes and collaborative exercises [163,199] and in multiplexed methylation -sensitive
restriction enzyme PCR assays [153]. Interpretation of the presence of such markers
should proceed with care as microorganisms can be detected on non-target body locations,
probably either from their high abundance and/or carry-over [104].
Unsurprisingly, vaginal samples and menstrual blood are unable to be distinguished
and share metagenomic profiles [196,200], and in one study, samples taken from the penis
the vaginal markers responded while no female DNA was detected [168].
In an extension from simply identifying bacteria representative of the oral cavity, it
was [201] recognized that identifying them could be used to distinguish between expirated
bloodstains (such as those forced by airflow out of the nose or mouth) and impact spatter,
providing what could be an additional crucial piece of information for an investigation
(see Section 1.3).
adolescent female (generally the presence of semen is not assessed through RNA typing
but microscopic analysis and differential extraction is used). Thus, in the Netherlands,
RNA body fluid typing is mostly applied in sexual assault cases to assess the presence of
vaginal cellular material.
In the 25 cases that assessed vaginal cellular material, 56 samples were analyzed
of which 19 resulted in vaginal cellular material detected (13 cases). In nine samples
(four cases), saliva was detected (one of these samples, a penile swab, was also positive
for vaginal cellular material; the other saliva-positive samples were all finger/nail dirt
samplings). The corresponding DNA profiles of 53 of these 56 samples were mixed
male/female profiles, while three showed no male (amelogenin Y) DNA and a full female
profile (samplings from paper tissue, condom, or finger). The male/female ratio at the
amelogenin locus in these mixed samples varied from 0.8 to 178, but this bore no relation
to whether vaginal cellular material was seen or not. This is not unexpected as these are
casework specimens, not ground truth data, and the female component may derive from
other cells, for example skin cells, which are no longer included in the NFI body fluid RNA
assay used in 2020.
within a case; all four cases involved samplings taken from clothing). In five cases, skeletal
muscle was detected (both cases that involved a knife, one of the cases involving a bullet
and two cases involving clothing: for these latter two cases, CNS was observed in stains
analyzed within the case as well). In one of the two cases analyzed for body fluids, besides
blood, saliva was found in a mist-like pattern indicating expirated blood (this case was
analyzed using a body fluid assay not yet carrying nasal mucosa markers).
The alternative scenarios put forward by the defense fall into three categories: once
the time of deposition was questioned (this was for the case in which expirated blood was
implied; the suspect was suggested to have visited the scene a day later when the victims
had already died). Five times it was suggested that another person was involved (twice
handling the knife, three times wearing the clothing that contained CNS material matching
the victim). Five times another explanation was given (the suspect shot at a car not at the
victim; the victim started shooting first so it was self-defense; the victim fell of the stairs
(for the case in which blood was found on a baseball bat); the injuries were self-inflicted
and the large puddles of blood on the bed sheets were staged by using animal or menstrual
blood (this was the second case analyzed by body fluid typing); the CNS and blood on the
clothing of the suspect are not shown to be the result of violence (no suggestion was given
how CNS did end up on trousers and a shirt).
For these cases, the alternative scenarios follow three of the four general scenarios
proposed in Section 1.1. These were: (1) ‘time of deposition’, (2) ‘another person performed
the crime’ and (3) ‘innocent explanation’; ‘secondary transfer’ was not proposed. This
makes sense, as in these cases, generous amounts of cellular material matching the victim(s)
were found for which it is more difficult to invoke secondary transfer.
as RNases are active during the mild lysis step unless an ample amount of strong RNase
inhibitors is used [104].
Other body fluids of which the presence may be questioned, are saliva and peripheral
blood. For blood, molecular analyses are mostly applied for discerning peripheral blood
and menstrual secretion. Saliva may be assessed in allegations of licking, which means
that often body locations are sampled (breasts, genitals, mouth), and mixed DNA profiles
are expected.
To assess allegations of anal penetration, the identification of a rectal mucosa marker
was described recently [213]. A rectal mucosa marker is to be preferred over markers for
fecal matter as rectal mucosa provides a more direct link to invasive activities. Finding a
marker specific for rectal mucosa is challenging since many body cavities and tubular or-
gans in the human body are lined by mucous membranes (for instance, along the respiratory,
digestive, and urogenital tract), an epithelial tissue that secretes mucus. Thus, specificity
of mucous membrane genes may be an issue, they may prove general mucosa markers or
cross-react with some other body fluid derived from a mucous membrane [98,214].
myelin) [227]. Whether an evidentiary stain carries predominantly grey or white matter,
will affect which markers can respond.
With body fluids, samplings may have a more even distribution of cell types, due to the
liquid constitution. These have different biological functions that affect their localization.
For example, red blood cells function within the blood (amongst others for carriage of
oxygen) while white blood cells act mostly outside of the bloodstream (immune system).
As a consequence, white blood cells crawl into the lymphatic system by a process denoted
extravasation [228]). The lymphatic system is an extensive drainage network and several
other forensically relevant body fluids may thereby carry white blood cells (e.g., nasal
secretion [228]).
It is crucial to understand the biological function and performance on other body
fluids or tissues for markers included in a cell typing assay.
expression of mRNAs may result in signals above the detection threshold for the assay,
especially when samples are over-amplified (by using too much input or too many am-
plification cycles). Technological imperfections can also cause non-specific signals like
remnants of DNA in RNA assays (this is only an issue when it is not possible to design
cDNA-specific amplicons with for instance one primer spanning an exon-exon boundary)
or incomplete bisulfite conversion in methylation-based assays. The most difficult form
of non-specificity is when markers cross-react with cell types other than the intended cell
type. This occurs more with body fluids than with organs. For instance, saliva, nasal
secretion and vaginal material are produced in body cavities (oral, nasal, vaginal) that
are all lined with mucous membranes. These can, as previously mentioned, give rise to
expression of the same mucous markers in these body fluids (denoted general mucosa
markers [98]). These non-specific responses can show inter-individual variation: mRNA
vaginal markers were detected in nasal secretions in many donors, with no relation to
gender or having a cold [104]. As with sensitivity, thorough validation with different
donors is highly important to understand marker performance.
Outside the human body, factors such as high humidity, UV radiation and/or high
temperature can invoke degradation of nucleic acids, especially when stains have not
fully dried. DNA is more resistant to hydrolysis than RNA, as the 2’-OH group can attack
the 3’-phosphate, leading to hydrolytic cleavage of the phosphodiester bond, which may
lead to samples providing a DNA but no or hardly an RNA profile [97]. Amplicons in
RNA assays are therefore generally shorter (preferably under ~150 base pairs) than in
DNA typing (up to ~450 base pairs). Intramolecular base pairing within RNA molecules
(not only G:C and A:U but also G:U) may result in secondary and tertiary structures that
provide more stability to certain regions, and amplicons targeting such stable RNA regions
(StaRs) [95,235] may enhance RNA profiling. DNA methylation assays have the advantage
of using the same nucleic acid as used for DNA profiling, so there is no difference in
stability (the methylation marks remain stable also in body fluids deposited outside a
human body [236]).
male donor to semen (if detected), but assumptions may need to made, for example, that
no saliva from either one of the donors is present. When choosing a methodology for
casework application, its suitability for mixed samples needs to be considered to enable
appropriate interpretation.
body fluid questioned) and lastly the RNA of the stain is sequenced to see what SNPs are
present in the transcribed mRNA to see if/ who of the donors matches this sequence.
This is a developing area with multiple challenges at hand: (1) SNPs can affect the
stability or transcription level of RNAs, so the genotypes at the DNA and RNA level may
not align (for instance one appears heterozygous at the DNA level but since the presence
of a SNP affects RNA stability or expression level, one appears homozygous at the RNA
level; (2) linked SNPs should be regarded according to their genetic phase (equivalent to
a microhaplotype), which may be complicated when different SNPs reside in different
amplicons; (3) the approach is only to be explored for samples in which all donors can be
assumed as there is no clear strategy/consensus yet on how to deal with unknown donors.
It is probably best to limit the approach initially to two-person mixtures.
CpG-SNPs (SNPs nearby CpGs indicative of cell types) have also been described [247,248].
Methods not depending on bisulfite conversion (such as MSRE) may be most practical
to use to detect these as the bisulfite conversion will affect all non-methylated cytosines
complicating SNP detection and primer design. A MS-HRM assay targeting DACT1 that
is hypomethylated in semen was used [249] to determine if it was possible to associate
semen with a DNA profile in a mixture. Some progress was made, and it was possible
to determine whether the semen compromised the majority, almost half, or was in the
minority of the components in a mixed fluid.
7. Further Considerations
7.1. Other Marker Types and Technologies That Have Been Explored
7.1.1. Non-Coding RNAs
Various non-coding RNAs have been explored as markers for body fluid and tis-
sue identification.
miRNAs are a class of small RNA molecules, 18–25 nucleotides in length, which are
involved in the regulation (repression) of mRNA translation and stability [69,71]. with
various forensic applications including body fluid identification [250].
Adult-specific roles for miRNAs (adult physiology, cancer development or suppres-
sion) have been described in various stem cell populations [251] and show continuous/
steady expression. This means these markers may be less specific for body fluid and tissue
identification than might be suggested by the highly specific expression patterns seen in
embryos [252]. Additionally, since human miRNAs generally interact through limited base
pairing of only a few bases in the 5’ part of the miRNA, a miRNA can bind to many targets.
Conversely, an mRNA may be targeted by several (different) miRNAs. For these reasons, a
key limitation of miRNAs is their specificity. Nonetheless, many studies have proposed
miRNAs for body fluids such as blood, saliva, menstrual blood and semen [253–257] and
body tissues including brain, liver, skeletal muscle and skin [258]. miRNAs are stable [259]
and have been shown to be detectable in forensic-like samples when treated to a range of
environmental conditions [218,260] after laundering [261].
Methods to analyze miRNAs primarily include miRNA expression profiles to iden-
tify candidate markers, followed by qPCR to test specificity [253,255,256,258,262]. Some
approaches include the extraction and analysis of miRNA and DNA together [263] and
multiplexed miRNA panels [264] able to discriminate between venous blood, menstrual
blood, semen, and saliva. High throughput sequencing of miRNAs from body fluid sam-
ples has also been used to identify new candidates [265]. Statistical methods and reference
(endogenous) miRNA markers [262] are needed to measure miRNA expression with qPCR.
Recently classification algorithms have been applied to the interpretation of miRNA profiles
as detection is not a simple yes–no answer [259].
The longest known class of small non-coding RNA are the piwi-interacting RNA (piRNA),
26–31 nucleotides in length. Their role is in the transcriptional and post-transcriptional si-
lencing of transposable elements which may be differentially expressed in different body
fluids making them suitable candidates for identifying body fluids (although transposon
silencing is foremost important in the germline where piRNAs were initially found [266]).
Genes 2021, 12, 1728 19 of 32
Two papers [267,268] describe the characterization by RNA sequencing of piRNA expres-
sion profiles and the selection of three piRNA markers (piR-hsa-27622, piR-hsa-1207 and
piR-hsa-27493) that distinguish between venous blood and menstrual blood and a further
two (piR-hsa-27493 and piR-hsa-26591) to distinguish between saliva and vaginal material,
implemented as TaqMan RT-qPCR assays.
Short interfering RNA (siRNA) are another class of small non-coding RNA with a role
in gene expression [269]. To date there are no reports of their use as markers in forensic
science, perhaps because their role is to interfere with the expression of specific genes, by
degrading RNA after transcription, rather than to promote the expression or modification
of specific transcripts.
Long non-coding RNAs (lncRNAs) are RNA transcripts not translated into proteins
that appear to be involved in the regulation of gene expression. Approximately 78%
of them are tissue-based compared to approximately 18% of mRNA transcripts. One
of the best studied is the X-inactive-based transcript (XIST) involved in X-chromosome
inactivation [270,271]. To date, XIST has been incorporated into an assay [272] to positively
identify male and female cell material in forensic samples with a second study [273] further
testing the assay on forensic type samples.
7.1.2. Proteome
All body fluids and tissues contain unique proteomes, derived from the expression of
mRNAs (see Section 3.1.1) in turn derived from the genome. In the past, unique proteins
of each body fluid or tissue type have been detected and presumptively identified by
determination of the enzyme activity (acid phosphatase for example) or immunological
properties (RSID tests (Independent Forensics, Lombard, IL, USA) for example). More
recently technological developments in proteomics, as for genomics, have highlighted
potential benefits for forensic identification purposes and useful reviews of the forensic
potential have been published [274–276].
Mass spectrometry (MS) is the analytical method of choice with advantages including
high sensitivity and specificity [277]. Generally, candidate markers are found by de novo
analysis or from reference proteomes, then validated by testing on known samples. By
detecting multiple peptides in a single assay from more than one protein per fluid, the assay
can be considered not only confirmatory but also human-based and suitable for mixtures.
Sensitive and specific assays have been developed including blood, semen, seminal fluid,
saliva, vaginal/ menstrual fluid and urine [277–280] using methods such as matrix-assisted
laser desorption/ionization (MALDI) MS [277], quadrupole time-of-flight MS [278,279]
MALDI MS Profiling and Imaging [281] and MALDI time-of-flight MS [282] from samples
as diverse as blood-stained finger marks [281] and blood and vaginal cellular material
under fingernails [282], although difficulties in identifying urine recovered from substrates
and semen samples that had been mixed with lubricants were found [280]. A challenge
to be addressed is that the protein content of each fluid/ tissue varies considerably, and
whilst it is possible to detect very small amounts of blood in saliva, the opposite was not
true [277–279].
MS methods tend to be destructive to the sample, however in a recent report [283]
a new approach, sheath-flow probe electrospray ionisation MS was used, which allows
stains to be sampled with no preparation or sample destruction bar the initial small amount
taken. In the research which examined blood, saliva and urine, alterations were observed
in the spectra over time suggesting that this approach could also be used to age a stain.
The relationship between the proteome and the genome offers the possibility for
proteomic “genotyping” [284,285] in a similar manner to the suggested RNA SNP and
CpG SNP approaches described in Section 6.4. This has clear advantages when DNA in a
sample is compromised in quality and quantity such as in a hair shaft, as proteins are more
stable and resistant to damage and decay than DNA. Genetic variations in human DNA
can be linked to mutations found in hair proteins (genetically variant peptides (GVPs)
referred to as single amino acid polymorphisms, SAPs, which in turn can be associated
Genes 2021, 12, 1728 20 of 32
with their DNA counterparts (missense SNPs). GVPs containing SAPs can be identified
by mapping these peptide sequences using MS and results have been obtained from short
lengths of human hair [275,286]. Not only could this approach be used to “identify” and
link individuals, but it can also be used to infer ancestry as SNP profiles varied between
ancestral groups.
7.1.3. Aptamers
Aptamers are single-stranded oligonucleotides or peptides that are selected using
SELEX (systematic evolution of ligands by exponential enrichment) in a sequential process
to bind their target molecules in a highly specific and sensitive manner. Initial studies used
RNA aptamers [287,288], with the first description of a DNA aptamer in 1992 [289] and
the first XNA aptamer capable of binding to a small molecule (ochratoxin A) in 2018 [290].
Aptamers have a number of advantages over assays such as the immunochromatogenic-
based methods described in Section 2.2.1. Consistent aptamer synthesis methods, more
straightforward functionalization, equivalent binding affinities and cheaper long term and
stable production are all factors.
Aptamers are now widely used as biosensors in a large number of fields [291,292]
with significant and recent examples seen for the rapid detection of SARS-CoV-2 [293,294]
and have potential for the use in forensic science [295]. Aptamers have been developed
to detect PSA [296], hemoglobin [297] and more recently, aptamers to detect spermatozoa
have been described using a combination of Cell-SELEX and massively parallel sequencing
technologies [295]. Once suitably specific and sensitive aptamers are obtained, they can be
built into a range of sensors for detection many of them suitable for field deployment.
8. Concluding Remarks
Preferably, body fluid typing and DNA profiling (to answer the what and the who
question) are analyzed on the exact same sample. This can be achieved when the DNA
extract is used for cell type inference (as with methylation analysis) or when RNA is
extracted from a fraction normally discarded during DNA extraction (like a flow through
fraction when binding DNA to a silica column). Case reports have been published for
both methylation [298] and RNA-based assays [299]. The advantage of using DNA for
body fluid typing is that no additional extraction procedure is needed; the disadvantage is
that less DNA remains for further analyses such as Y-STR profiling, mtDNA analysis or
the prediction of age, ancestry, appearance. Microbiome analysis can use either DNA or
RNA. The global COVID-19 pandemic has stimulated the development of assays that may
find their use in forensic analysis such as multiplexed and extraction-free amplification
assays [300], which may provide RNA-based tests prior to DNA extraction and analysis
that may present the opportunity of triage. Such extraction-free methods [301] present
pioneering opportunities beyond advancements such as one-step RT-PCR methods that
simplify current methodology [302].
Introducing cell typing methodologies in a forensic laboratory requires extensive
validation, implementation of interpretation and reporting, and explaining results for
judiciary. This comes with additional costs (also because the technique itself may ask quite
some hands-on laboratory work) and it has been suggested that it is best performed or
outsourced to specialized laboratories.
Author Contributions: S.H. and T.S. both wrote sections of the original draft (S.H. Sections 2, 3 and 7;
T.S. Sections 1 and 4–6). Reviewing and editing was performed by both S.H. and T.S. The authors
apologize that not all literature could be referenced. All authors have read and agreed to the published
version of the manuscript.
Funding: This research received no external funding.
Acknowledgments: We thank Margreet van den Berge (NFI) and Stephanie Opperman (ESR) for
critical reading of parts of the manuscript.
Conflicts of Interest: The authors declare no conflict of interest.
Genes 2021, 12, 1728 21 of 32
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